豌豆蚜温度受体基因Painless的克隆及时间和组织表达

Cloning and Spatio-Temporal Expression of the Thermoreceptor Gene Painless in Pea Aphids(Acyrthosiphon pisum)

【目的】从豌豆蚜(Acyrthosiphon pisum)触角中克隆候选的温度受体基因——Painless,分析该基因所编码的蛋白结构特点,研究该基因在昆虫不同发育时期和不同组织中的表达谱,探讨该基因的功能。【方法】通过生物信息学的方法,使用果蝇(Drosophila melanogaster)Painless从豌豆蚜基因组数据库AphidBase中预测ApisPainless的假定全长序列,之后根据预测得到的假定全长序列,使用Primer premier 5.0设计全长引物,使用RT-PCR方法从豌豆蚜触角cDNA中克隆ApisPainless的全长序列,并应用DNAMAN软件对预测序列与克隆序列进行比对,确定克隆序列与预测序列一致后,使用在线工具SMART(simple modular architecture research tool)对克隆得到的ApisPainless蛋白结构进行预测,分析其序列N端的锚蛋白重复序列及跨膜域,通过使用在线比对软件Clustal Omega对ApisPainless与DmelPainless的3个可变剪切型(isoform A、B、C)进行多序列比对,分析ApisPainless的可变剪切类型,通过构建昆虫TRP(transient receptor potential)离子通道超家族的进化树,分析ApisPainless的进化地位,并通过绘图直观地比较TRPA亚家族4个成员TRPA1、Painless、Pyrexia和Water witch的序列长短及N端锚蛋白重复序列的差异,使用实时定量荧光PCR技术,研究ApisPainless在1—4龄若虫以及成虫这5个不同发育时期的相对表达量,以及其在豌豆蚜成虫的触角、头、足和胸腹这4个不同组织中的相对表达量。【结果】预测并克隆得到ApisPainless全长序列,该基因的全长序列为2 832 bp,编码943个氨基酸,蛋白结构分析表明ApisPainless具有8个锚蛋白重复序列以及6次跨膜结构,通过序列比较发现ApisPainless与DmelPainless isform A的相似性最高,达到42.3%。进化树分析结果表明昆虫TRP超家族分为TRPA、TRPC、TRPM、TRPN、TRPV、TRPP和TRPML 7个亚家族,而ApisPainless聚类于TRPA亚家族Painless一支,在TRPA亚家族TRPA1、Painless、Pyrexia和Water witch这4个成员中,TRPA1的序列最长,约为1 200个氨基酸,其后依次是Water witch、Pyrexia和Painless,氨基酸数目约有920—1 000个。从N端的锚蛋白重复序列上看,Water witch和Pyrexia约有9个重复序列,Painless约为8个,而TRPA1则高达15—16个,发育时期表达谱分析结果显示ApisPainless在豌豆蚜的各个发育阶段均有表达,其中以1龄若虫期表达量最高。组织表达谱分析结果显示,ApisPainless在豌豆蚜的触角、头、足和胸腹各组织中均有表达,其中以在触角中的表达量最高,足中的表达量居其次。【结论】克隆得到的ApisPainless是TRPA亚家族Painlss基因,其在触角及足中表达量高,推测其功能可能类似于果蝇的Painless,参与高温伤害识别、机械刺激识别及味觉探测等过程。

【Objective】 The objective of this study is to clone a candidate thermoreceptor gene Painless in the antenna of pea aphids, Acyrthosiphon pisum, and to illustrate the protein structure encoded by this gene, further to characterize the expression profiles of this gene across developmental stages and in different tissues. 【Method】 Based on the sequence of Painless from Drosophila melanogaster, full-length sequence of the ApisPainless from AphidBase was predicted by bioinformatic analysis, and the gene was cloned with cDNA template obtained from pea aphid antennae and specific primers were designed by Primer premier 5.0 software using RT-PCR. After determining the consistency of cloned sequence with predicted one by DNAMAN software, the protein structure of ApisPainless was depicted through the online tool SMART（simple modular architecture research tool） according to the analysis of ankyrin repeats at N-terminus and transmembrane domains. Subsequently, the Clustal Omega software was introduced to conduct alignment and analysis of ApisPainless with DmelPainless isforms（A, B and C）. The phylogenetic tree was constructed for the analysis of insect TRP（transient receptor potential） channel superfamily, especially for the subfamily TRPA, which includes 4 members of TRPA1, Painless（ApisPainless）, Pyrexia and Water witch, by comparing the length of their protein sequences and ankyrin repeats at N-terminus. Quantitative real-time PCR was applied to characterize the relative expression levels of ApisPainless at different developmental stages（1st-4th instar nymphs and adults） and in different tissues（antennae, heads, legs, and thoraxes and abdomens）. 【Result】 The ApisPainless was successfully predicted and cloned, with a complete CDS of 2 832 bp, encoding 943 amino acids. Protein structure analysis indicated the ApisPainless had 8 ankyrin repeats and 6 transmembrane domains. The ApisPainless was more similar to the DmelPainless isoform A with the similarity of 42.3%, and clustered to the insect Painless cluster based on the results of multiple sequence alignment and phylogenetic analysis, respectively. TRPA1 was composed of about 1 200 amino acids, making it the longest among the TRPA subfamily, followed by Water witch, Pyrexia and Painless, with 920-1 000 amino acids. In addition, there were 8 ankyrin repeats observed at N-terminus in Painless, while 9 in both Water witch and Pyrexia, and 15-16 in TRPA1. ApisPainless was expressed at the highest level in the 1st instar nymphs, even though it was universally expressed across all the stages. Tissue-specific expression analysis elucidated that ApisPainless was expressed in a whole body-expressing manner, but mostly expressed in antennae, followed by legs. 【Conclusion】ApisPainless obtained in this study was clustered to Painless, which is a member of insect TRPA subfamily. The ApisPainless is highly expressed in antennae and legs of pea aphids, which implies that it may be involved in noxious heat sensation, mechanosensation and gustatory detection as its homolog Painless in D. melanogaster.